Osteoclast differentiation depends on support provided by stromal cells. Although indirect evidence suggests that these stromal cells may be of the osteoblast lineage, the identity of these cells and their relationship to matrix-synthesizing osteoblasts remains unknown. One of the key mechanisms by which stromal cells support osteoclast differentiation is expression of receptor activator of NF(B ligand (RANKL). Hormones that stimulate osteoclast formation, such as PTH, do so by stimulating RANKL expression in stromal cells. However, the mechanisms controlling RANKL expression in these cells are only partially understood. The goals of this application are to elucidate the molecular mechanisms that control RANKL expression in stromal cells and determine the relationship, if any, of these cells to matrix-synthesizing osteoblasts. During the previous funding cycle, we demonstrated that PTH controls RANKL expression in stromal cells via a distant transcriptional enhancer that we have designated the RANKL distal control region (DCR). DCR knockout mice have reduced RANKL expression and increased bone mass and strength. The transcription factor Runx2 binds to the DCR, suggesting a linkage to osteoblast differentiation; however, stimulation of RANKL was not altered in cells from Runx2-deficient mice. Therefore, the molecular basis of DCR action and RANKL cell type-specific expression remains unclear. We also determined that ablation of matrix-synthesizing osteoblasts and their immediate precursors did not alter RANKL expression or osteoclast formation in bone, highlighting the contention that the identity of stromal cells remains unknown. Based on these findings, we hypothesize that matrix-synthesizing osteoblasts and the stromal cells that support osteoclastogenesis are derived from a common mesenchymal precursor, which bifurcates early in its specification into these two distinct lineages. Aim 1 will identify the molecular mechanisms by which the DCR functions by determining whether other members of the Runx family of transcription factors are required for RANKL expression in the absence of Runx2. In addition, other proteins that bind the DCR will be identified by footprinting and their role in DCR action determined by gain- and loss-of-function studies. Aim 2 will identify osteoclast support cells using two complementary approaches: (1) by localizing RANKL-expressing cells and their descendants in bone and (2) by determining whether RANKL expression in genetically defined cell populations is required for osteoclast formation. PUBLIC HEALTH RELEVANCE: The proposed studies seek to identify stromal cells that support production of bone-degrading cells. This will be accomplished by studying a gene expressed in stromal cells that is essential for production of bone-degrading cells. Increased understanding of the mechanisms that control bone resorption will provide essential information for the development of therapies to maintain or increase bone mass and strength, thereby reducing the risk of osteoporotic fractures.